The RPE is a monolayer of pigmented cells that interact with retinal photoreceptors and helps maintain visual function. We have developed a new preparation to study RPE physiology in rat that includes the neural retina. The retina-retinal pigment epithelium-choroid (R-RPE-Ch) preparation was mounted in an Ussing chamber which allowed independent perfusion of retinal and choroidal baths. The intact monolayer had a mean (+/- SD) transepithelial potential of 4.3 +/- 1.0 mV and a total transepithelial resistance of 152 +/- 37 ohm*cm-squared (n =10). As in other mammalian RPE preparations, we found barium-sensitive potassium channels at the apical and basolateral membranes. Intracellular microelectrodes at the basolateral membrane showed that step increases of basal potassium concentration depolarized the basolateral membrane 8 mV (n=2) and were blocked by barium. Decreasing apical potassium concentration mimics light-onset and hyperpolarizes the apical membrane;increasing apical potassium concentration depolarized the apical membrane by 6 mV (n=3). The potassium concentration responses were blocked by barium. Elevation of calcium concentration in the basal bath 10 fold, from 1.8 to 18 mMincreased TTP by 0.6 mV(p<0.001.;n=5). This increase was inhibited by 50% (p<0.01;n=3) when pretreated with DIDS (1mM) on the basolateral side. The R-RPE-Ch preparation was developed to study light-induced changes in RPE physiology, and provides an in-vitro model of in-vivo function. It can be used to analyze light-evoked alterations in RPE physiology that occur in Bests disease, which is thought to result from a mutation in basolateral membrane calcium-activated chloride channels.